Digoxin is widely used in the treatment of cardiac irregularities. Efficacy of a digoxin dose, as administered to a patient, is dependent on many factors. A narrow therapeutic index necessitates an accurate and reliable means for the measurement of serum digoxin concentration. The measurement for digoxin concentrations in the serum is complicated by the metabolites of digoxin in the serum. There is considerable variation between patients regarding the manner in which digoxin is metabolized in vivo. Digoxin is a steroid-carbohydrate conjugate. A major route of metabolism is the sequential loss of glycose units and/or saturation of the steroid lactone ring. The resulting metabolites retain various degrees of biological and toxic activities of the native digoxin. The presence of metabolites have prevented the accurate measurement of digoxin by standard radioimmunoassay techniques. Serum levels of digoxin have been routinely measured by immunoassay using an antidigoxin antibody raised against a bovine serum albumin-digoxin conjugate immunogen. As described in Butler et al, (1967) Digoxin Specific Antibodies, Proc. Natl. Acad. Sci, 57:71-78, the bovine serum albumin (BSA)-digoxin conjugate is prepared by periodate oxidation of the vicinal hydroxyyl groups of the terminal sugar. The generated aldehyde groups are coupled to the amino groups of BSA. Thus the conjugate linkage is through the carbohydrate moiety of digoxin. The antibodies generated in vivo against this conjugate are, therefore, for the most part directed against the steroid moiety of digoxin. Thus, the metabolites, such as digoxigenin bis and monodigitoxide and digoxigenin all react with the antibodies whilst other metabolites such as dihydrodigoxigenin and dihydrodigoxin, where the C22 carbon is reduced, show little or no cross-reactivity with the antibodies. The cross-reactivity of the metabolites of digoxin with the antidigoxin antibodies is an appreciated fact and the specifications of most commercial antidigoxin sera state the degree of cross-reactivity. It has been found that for some antisera, the carbohydrate metabolites are more potent antigens than digoxin itself, the additional carbohydrate units in some way reducing the antibody binding activity of native digoxin. As a result, there is considerable variation in the anticipated extent to which the glycosidic metabolites may account for the measured digoxin concentration in serum samples.
As reported in Soldin, S; Papanastasion-Diamand, A; Heyes, J; Lingwood, C. A. and Olley, P, (1984) Are Immonuassays for Digoxin Reliable? Clin Biochem, 17 317-320, a detailed study of the specificity of the radioimmune assay for digoxin reveals that, in addition to the cross-reactivity of the metabolites, some thirty additional cross-reactive, often unrelated compounds have been identified in various serums.